Method of treating cancer using adenosine and its analogs

ABSTRACT

The present invention provides methods of treating individuals having malignancies associated with estrogen receptor activity comprising administering to an individual affected with said malignancy, an effective amount of adenosine analog in a pharmaceutical carrier to downregulate or diminish estrogen receptors in the cells. The invention further provides methods of identifying novel adenosine analogues capable of treating malignant cells expressing estrogen receptors. The invention also provides kits comprising adenosine analogs for downregulating estrogen receptors in cells and kits for screening for novel adenosine analogs capable of downregulating estrogen receptors. Further, the invention provides uses of adenosine analogs in downregulation of estrogen receptors, cell growth and cell cycle, as well as pharmaceutical compositions comprising adenosine analogs effective in suppressing cellular growth, cell cycle or downregulating estrogen receptors.

This application claims benefit under 35 U.S.C. §119(e) of 60/414,706filed Sep. 30, 2002.

This invention was supported by National Institutes of Health grant No.CA79397 and the government of the United States has certain rightsthereto.

FIELD OF THE INVENTION

The present invention is directed to a method of treatingestrogen-receptor positive cancers comprising administering to anindividual in need thereof adenosine receptor agonists that are capableof downregulating estrogen receptors. Preferably the cancer is breastcancer.

BACKGROUND OF THE INVENTION

The human estrogen receptor (ER) is a member of the nuclear receptorsuperfamily of transcription factors (Evans, Science 240:889-895(1988)). Upon binding a ligand, ER undergoes a conformational changeinitiating a cascade of events ultimately leading to its associationwith specific regulatory regions within target genes (O'Malley et al.,Hormone Research 47:1-26 (1991)). The ensuing effect on transcription isinfluenced by the cell and promoter context of the DNA-bound receptor(Tora et al. Cell 59:471-487 (1989), Tasset et al., Cell 62:1177-1181(1990); McDonnell et al. Mol. Endocrinol. 9:659-669 (1995); Tzukerman etal. Mol. Endocrinol. 8:21-30 (1994)). It is in this manner that thephysiological ER-agonist, estradiol, exerts its biological activity inthe reproductive, skeletal and cardiovascular systems (Clark and Peck,Female Sex Steroids:Receptors and Function (eds) MonographsSpringer-Verlag, New York (1979); Chow et al., J. Clin. Invest. 89:74-78(1992); Eaker et al. Circulation 88:1999-2009 (1993)).

Approximately 180,000 women are diagnosed with breast cancer each yearin the United States. Most of these women are treated using surgery andlocal radiotherapy. However, nearly 60,000 women still go on to developmetastatic breast cancer each year, and about 45,000 of these patientseventually die from their malignancies. While metastatic breast canceris rarely curable, it is treatable with modern pharmaceuticals that canprolong patient survival and reduce the morbidity associated withmetastatic lesions. Foremost among these therapies are hormonalmanipulations that include selective estrogen receptor modifiers(SERMs). SERMs are small ligands of the estrogen receptor that arecapable of inducing a wide variety of conformational changes in thereceptor and thereby eliciting a variety of distinct biologicalprofiles. SERMs not only affect the growth of breast cancer tissue butalso influence other physiological processes. The most widely used SERMin breast cancer is tamoxifen, which is a partial estrogen receptoragonist/antagonist that produces objective responses in approximately50% of the patients. Unfortunately, almost all patients who taketamoxifen eventually relapse with tamoxifen-resistant tumors.Approximately half of the patients who fail tamoxifen treatment willrespond to a subsequent hormonal manipulation therapy such asovariectomy, aromatase inhibitors, or other SERMs. The second linetherapies for hormonal manipulation therapy of metastatic breast cancerrepresent a substantial unmet need because no single agent has becomethe treatment of choice for patients who fail tamoxifen therapy. Theideal agent would be a medication that induces regression of metastaticbreast cancer lesions in women who have previously responded totamixofen therapy.

SERMs modulate the proliferation of uterine tissue, skeletal bonedensity, and cardiovascular health, including plasma cholesterol levels.In general, estrogen stimulates breast and endometrial tissueproliferation, enhances bone density, and lowers plasma cholesterol.Many SERMs are bifunctional in that they antagonize some of thesefunctions while stimulating others. For example, tamoxifen, which is apartial agonist/antagonist of estrogen receptor, inhibitsestrogen-induced breast cancer cell proliferation but stimulatesendometrial tissue growth and prevents bone loss.

Estrogen has also been shown to function as a mitogen inestrogen-receptor (ER) positive breast cancer cells. Thus, treatmentregiments which include antiestrogens, synthetic compounds which oppose,the actions of estrogen have been effective clinically in halting ordelaying the progression of the disease (Jordan and Murphy, EndocrineReviews 11:578-610 1990); Parker, Breast Cancer Res. Treat. 26:131-137(1993)).

One of the most studied estrogen receptor function interfering compoundsis tamoxifen (TAM), (Z)1,2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene, (Jordan and Murphy, Endocrine Reviews11:578-610 (1990)). As discussed above, tamoxifen functions as anantagonist in most ER-positive tumors of the breast and ovum, butdisplays a paradoxical agonist activity in bone and the cardiovascularsystem and partial agonist activity in the uterus (Kedar et al. Lancet343:1318-1321 (1994); Love et al., New Engl. J. Med. 326:852-856 (1992);Love et al., Ann. Intern. Med. 115:860-864 (1991)). Thus, theagonist/antagonist activity of the ER-tamoxifen complex is influenced bycell context. This important observation is in apparent contradiction tolongstanding models that hold that ER only exists in the cell in anactive or an inactive state (Clark and Peck, Female SexSteroids:Receptors and Functions (eds) Monographs on Endocrinology,Springer-Verlag, New York (1979)). Rather it indicates that differentligands acting through the same receptor can have different biologicaleffects in different cells. Definition of the mechanism of thisselectivity is likely to advance the understanding of processes such astamoxifen resistance, observed in most ER-containing breast cancers,where abnormalities in ER-signaling are implicated (Tonetti and Jordan,Anti-Cancer Drugs 6:498-507 (1995)).

Tamoxifen, as well as a structurally similar compound known as4-OH-tamoxifen, raloxifene, and ICI 164,384 have been developed for thetreatment and/or prevention of osteoporosis, cardiovascular disease andbreast cancer in addition to the treatment and/or prevention of avariety of other disease states. Both compounds have been shown toexhibit an osteoprotective effect on bone mineral density combined witha positive effect on plasma cholesterol levels and a greatly reducedincidence of breast and uterine cancer. Unfortunately, tamoxifen andraloxifene both have unacceptable levels of life-threatening sideeffects such as endometrial cancer and hepatocellular carcinoma.Therefore, there is a need for new breast cancer therapies.

SUMMARY OF THE INVENTION

It is therefore the purpose of the present invention to provide a novelmethod for treating individuals affected with cancers associated withestrogen receptor expression, such as estrogen receptor positivecancers, including breast and ovarian cancers.

In one embodiment, the invention provides a method of treating breastcancer in an individual in need thereof by administering an effectiveamount of at least one adenosine analog and a pharmaceuticallyacceptable carrier to decrease estrogen receptors.

Estrogen receptors according to the present invention include estrogenreceptor alpha and estrogen receptor beta. In one preferred embodiment,the estrogen receptor is estrogen receptor alpha.

The purine nucleoside adenosine is a natural metabolite that plays arole in several physiologic and pathologic processes, such as inhibitionof platelet aggregation, cardioprotection after ischemia, vasodilation,mast cell activation and lypolysis (see review (1)). Adenosine isproduced and released at micromolar concentration in/from severaltissues, such as fibroblasts, endothelial cells, epithelial cells,cardiac myocytes, muscle cells, and platelets (2-5). The level ofadenosine is further elevated under conditions such as muscle exercise(6), or ischemia (7).

Adenosine exerts many of its effects by activation of specific cellsurface receptors. To date, four adenosine receptors (AR), the A1AR,A2aAR, A2bAR and A3AR have been cloned (8, 9). Medicinal chemistry hasprovided different adenosine analogs that are potent selectiveactivators of specific adenosine receptors. These include agonists, suchas 2-Chloro-N⁶-cyclopentyladenosine (CCPA) (A1AR selective),2-p-(2-Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosineCGS-21680 (A2aAR selective),N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) (A3ARselective) and 5′-(N-Ethylcarboxamido)adenosine (NECA) (activates bothA2aAR and A2bAR).

Adenosine and its analogues were recently shown to inhibit growth orinduce apoptosis in several types of cancer cells. Epidermoid carcinomaA431 cells and some human cancer cells were inhibited by agonists forA1AR or A2AR (10-12). HL-60 leukemia and U-937 lymphoma cells werereported to be induced into apoptosis by A3AR agonists (13, 14). Fishmanet al found that adenosine is one active component within skeletalmuscle cell-conditioned medium, which can inhibit the growth of SK-28melanoma cells, K-562 chronic myelogenous leukemia cells, and MCF-7breast cancer cells (15).

Preferably, the estrogen receptor down-regulating adenosine analog orderivative thereof is selective to the A3 adenosine receptor (A3AR). Inone preferred embodiment, the adenosine analog is selected from a groupconsisting of N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide(IB-MECA), 2-chloro-deoxyadenosine (CdA), 3′-deoxyadenosine(Cordycepin), 2-chloro-N-6-cyclopentyladenosine (CCPA),5′-(N-Ethylcarboxamido) adenosine (NECA), 2-chloro-adenosine (CADO),inosine (INO) or a derivative or a combination thereof.

In one preferred embodiment, the adenosine analog useful according tothe present invention is IB-MECA, CdA, Cordycepin or a derivative or acombination thereof.

In the most preferred embodiment, the estrogen receptor down-regulatingadenosine analog is IB-MECA or a functional, estrogen receptordown-regulating derivative thereof. Preferably, the estrogen receptor isestrogen receptor alpha.

Estrogen receptors are known to be expressed in various human tissuesincluding reproductive tissues such as ovaries, uterine, vagina, andtesticles (for review, see, e.g. OMIM athttp://www.ncbi.nlm.nih.gov/entrez). These receptors are also present insome pituitary adenomas and osteosarcomas. The estrogen receptorexpression in mammary glands and their relationship with breast cancerhas been widely studied.

Two isoforms of human estrogen receptor, ER-alpha (ESRA, OMIM ID. No.133430; GenBank ID Nos. gi:182192 and gi:31233) and ER-beta (ESRB, OMIMID No. 601663, GenBank ID Nos. gi:2911151 and gi:34193698), have adistinct, although sometimes overlapping expression pattern. Further,additional ESR isoforms, generated by alternative mRNA splicing, havebeen defined in several tissues and they are postulated to play a rolein tumorigenesis or in modulating the estrogen response (OMIM entry No.601663, at http://www.ncbi.nlm.nih.gov/entrez). The present inventioncontemplates downregulating estrogen receptors in general. In onepreferred embodiment, the estrogen receptor is estrogen receptor alpha.

An individual in need of treatment may have any malignancy which isassociated with estrogen receptor mediated growth. Such malignanciesinclude, but are not limited to breast tumors, osteosarcomas (Chaidarun,et al., Molec. Endocr. 12: 1355-1366, 1998), pituitary adenomas(Shupnik, et al., J. Clin. Endocr. Metab. 83: 3965-3972, 1998) as wellas cancers of human reproductive organs expressing estrogen receptorsincluding ovaries, uterus, and testicles, particularly in the Leydigcells.

In one preferred embodiment of the present invention, the adenosineanalog down-regulates estrogen receptor levels in the transcript level.Therefore, the invention is particularly useful in treating malignancieswhich are caused by mutated and/or truncated estrogen receptors thatactivate transcription even in the absence of estrogen, and cannottherefore be inhibited with pharmaceutical compounds functioning asestrogen analogs.

The estrogen receptor down-regulating analogue according to the presentinvention also includes mixtures of different estrogen receptordown-regulating analogues.

In a preferred embodiment, the individual in need of treatment byadenosine analogs is affected with an estrogen receptor alpha (ERalpha)positive cancer, such as breast cancer including ductal carcinoma insitu (DCIS), infiltrating (or invasive) ductal carcinoma (IDC), orinfiltrating (or invasive) lobular carcinoma (ILC).

Examples of ERalpha positive cells useful according to the presentinvention include, but are not limited to breast cancer cell (BCC) linesincluding but not limited to MCF-7 (high amount), T-47D, ZR-75, CAMA-1,BT483, BT474, MDA-MB-361, and MDA-MB-134.

Non-exclusive examples of estrogen receptor beta positive cells includebreast tumor cells, ovarian tumor cells (Chu, S. et al., Estrogenreceptor isoform gene expression in ovarian stromal and epithelialtumors. J. Clin. Endocr. Metab. 85: 1200-1205, 2000), and pituitaryadenomas including prolactinomas, mixed growth hormone/prolactinetumors, gonadotroph tumors, and somatotroph, corticotroph, and null celltumors (Chaidarun, S. S. et al., Differential expression of estrogenreceptor-beta (ER-beta) in human pituitary tumors: functionalinteractions with ER-alpha and a tumor-specific splice variant. J. Clin.Endocr. Metab. 83: 3308-3315, 1998).

Further, any malignant cell type which can be shown to express estrogenreceptors using either protein or mRNA expression, using method wellknown to one skilled in the art, is considered to be a target malignancyfor the methods of the present invention.

In one embodiment, the method of the present invention comprisesadministering ERalpha down-regulating agonists before, after orsimultaneously with tamoxifen ((Z)1,2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene), 4-OH-tamoxifen(4-OH-(Z)1,2-diphenyl-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1-butene),raloxifene, and ICI 164,384(N-(n-butyl)-11-[3,17β-dihydroxyestra-1,3,5(10)-trien-7α-yl]N-methylundecanamide).

In one embodiment, the invention provides a method of treating breastcancer with an estrogen receptor alpha mutation Tyr 537 to Asn (T 1609A), by administering an estrogen receptor down-regulating amount of anadenosine analog to the individual with cells having the mutation. Thismutation has been identified in approximately 1 of 30 metastatic breastcancers (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=133430).This substitution confers constitutive transcriptional activity toestrogen receptor and its activity cannot be antagonized withantiestrogens such as tamoxifen and pure antiestrogen ICI 164384 (ZhangQ. X. et al., Cancer Res., 1997, April 1; 57(7):1244-9).

In one embodiment, the invention provides a method of identifying novelcompounds useful for down-regulating estrogen receptors. In this way,one can identify compounds, including adenosine analogs and derivativesthereof, useful for treating estrogen-receptor positive cancers. Themethod comprises the steps of contacting an ERalpha or estrogen receptorbeta (ERbeta) positive cell with a test compound and calculating cellgrowth, measuring ERalpha or ERbeta levels by western blot analysisand/or quantitative RT-PCR, and determining cell cycle arrest by flowcytometry analysis. Cells and cell lines useful according to thisembodiment include cell lines expressing ERalpha, such as MCF-7, T-47D,ZR-75, CAMA-1, BT483, BT474, MDA-MB-361, and MDA-MB-134.

In one preferred embodiment, the method comprises administering a testcompound to cells and detecting the level of ER transcripts from thecells. If the ER transcript level is decreased compared to the samecells grown in the absence of the test compound, the test compound isconsidered to have an ER down-regulating activity. In one embodiment theER is ERalpha. In an alternative method the ER is ERbeta.

The invention further provides kits for downregulating estrogenreceptors, kits for detecting novel estrogen receptor downregulatingadenosine analogs, and uses to of adenosine analogs to downregulateestrogen receptors, cell growth and cell cycle, and pharmaceuticalcompositions comprising adenosine analogs to downregulate cell growth,cell cycle and/or estrogen receptor level in the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of adenosine and adenosine analogs.

FIGS. 2A-2E show the effects of adenosine and adenosine receptoragonists on MCF-7 cell colony formation. MCF-7 cells were plated in softagar and treated with adenosine (FIG. 1A), CCPA (FIG. 1B), CGS21680(FIG. 1C), NECA (FIG. 1D), or IB-MECA (FIG. 1E), with the indicatedconcentrations. After two weeks of treatment, colony numbers werecounted and expressed as the percentage of those of vehicle-treatedcells (0 μM). Data shown are averages of triplicate experiments anderror bars represent standard deviations.

FIGS. 3A-3C show the effect of IB-MECA on colony formation, growth andapoptosis of different breast cancer cell lines. In FIG. 3A, humancancer cell lines MCF-7, ZR-75, T47D, Hs578T and HeLa were plated insoft agar and treated with 100 μM IB-MECA. Numbers of colonies formedwere determined after two weeks in culture, and expressed as thepercentage of those of vehicle-treated cells (DMSO). In FIG. 3B, MCF-7,ZR-75, T47D and Hs578T cells were plated in 6 well plates, and treatedwith 100 μM of IB-MECA for three days. Cell numbers were counted andexpressed as percentages of cell counts before treatment (Day 0). InFIG. 3C, MCF-7, ZR-75, T47D and Hs578T cells were treated with 100 μMIB-MECA for two days. Cells were stained with propidium iodide andsubjected to FACS analyses. Apoptotic events were determined byquantification of the sub-2n populations on fluorescence histograms, andwere expressed as the percentage of total events. All data shown areaverages of triplicate experiments, and error bars represent standarddeviations.

FIGS. 4A-4D show that IB-MECA induces growth inhibition anddownregulates cyclins in MCF-7 cells. In FIG. 4A, MCF-7 cells weretreated with vehicle (DMSO) or 100 μM IB-MECA and were counted after 1,2 or 3 days. The number of cells was expressed as the percentage of cellcount before treatment (Day 0). Data shown are averages of triplicateexperiments and error bars represent standard deviations. In FIG. 4B,MCF-7 cells were treated with vehicle (DMSO) or 100 μM IB-MECA for 2days. Cells were stained with propidium iodide (PI) and subjected toFACS analyses. The percentages of cells in different phases of the cellcycle were as follows: G1 phase: 50.2% (DMSO) and 64.2% (IB-MECA); Sphase: 25.2% (DMSO) and 12.3% (IB-MECA); G2/M: 24.6% (DMSO) and 23.4%(IB-MECA). These calculations represent averages of 3 determinations.Representative fluorescence histograms are shown. In FIG. 4C, MCF-7cells were treated with 100 μM IB-MECA for the indicated times. Cellswere harvested and subjected to Western blot analyses using indicatedantibodies. In FIG. 4D, MCF-7 cells were treated with different dosagesof IB-MECA or NECA and harvested after 48 hours. Cells were subjected toWestern blot analyses using indicated antibodies.

FIGS. 5A and 5B show that the effect of IB-MECA is not throughactivation of the A3 adenosine receptor. MCF-7 cells were stablytransfected with human A3 adenosine receptor cDNA. In FIG. 5A, theexpression of A3 adenosine receptor in MCF-7 cells or a pool of stablytransfected cells (MCF-7+A3) was assayed by RT-PCR. Reversetranscription reactions were performed with (+) or without (−) reversetranscriptase, followed by PCR reactions using primers specific for A3adenosine receptor (A3AR) or GAPDH. Representative agarose gel picturesare shown. In FIG. 5B, MCF-7 cells or pool of MCF-7 cells stablyexpressing A3 adenosine receptor (MCF-7+A3) were plated into soft agarand treated with different concentrations of IB-MECA. Colony numberswere determined after two weeks in culture and expressed as those ofvehicle-treated cells. Data shown are averages of triplicate experimentsand error bars represent standard deviations.

FIGS. 6A-6D show how that IB-MECA treatment downregulates estrogenreceptor α mRNA level, protein level and transcriptional activity inMCF-7 cells. In FIG. 6A, MCF-7 cells were treated with vehicle (−) or100 μM IB-MECA (+) for the indicated times. Reverse transcriptionreactions were carried out with (+RT) or without (−RT) reversetranscriptase, using RNA isolated from the samples. Primers specific forestrogen receptor α and GAPDH were used in semi-quantitative PCRreactions. Pictures of RT-PCR products analyzed on agarose gels areshown. In FIG. 6B, estrogen receptor α (ERα), cyclins and actin (loadingcontrol) were assayed with Western blot analyses, using indicatedantibodies, after MCF-7 cells were treated with 100 μM IB-MECA for theindicated times. In FIG. 6C, MCF-7 cells were treated with differentconcentrations of IB-MECA or NECA for two days. Cells were harvested andsubjected to Western blot analyses with antibodies against ERα or actin.In FIG. 6D, MCF-7 cells transfected with pERE-Tk-Luc or pCMV-β-Galplasmids were treated with vehicle (O) or indicated concentrations ofIB-MECA for 12 hours. Cells were harvested and reporter gene activitywas assayed as detailed in Methods. Data shown are averages oftriplicate experiments and error bars represent standard deviations.

FIGS. 7A and 7B show that overexpression of estrogen receptor a rescuesgrowth inhibition by IB-MECA in MCF-7 cells. MCF-7 cells weretransiently transfected with pcDNA3-ERα (pERα) or pcDNA3 (vector) with atransfection efficiency of approximately 40% (see Methods). Cells weretreated with vehicle (DMSO) or 100 μM IB-MECA for one day. In FIG. 7A,expression of estrogen receptor α (ERα) was determined by Western blotanalysis. Actin served as a loading control. In FIG. 7B, cell numberswere determined post one day incubation, and expressed as the percentageof cell count before treatment (Day 0). Data represent averages oftriplicate experiments and error bars represent standard deviations.Samples labeled with “*” showed a p value of less than 0.002 underStudent's t-test.

FIGS. 8A-8E show the effects of IB-MECA on mRNA level and mRNA half-lifeof estrogen receptor α in MCF-7 cells. FIG. 8A shows the mRNA levels ofestrogen receptor a (ERα), pS2 and estrogen receptor β (ERβ) in IB-MECAtreated cells. MCF-7 cells were treated with vehicle (−) or 100 μMIB-MECA (+) for the indicated periods. Reverse transcription reactionswere carried out on total RNA isolated from the samples (same samples asin FIG. 6B). Primers specific for ERα, pS2, ERβ and GAPDH were used insemi-quantitative PCR reactions. Representative pictures of RT-PCRproducts analyzed on poly-acrylamide gels are shown. FIG. 8B showsresults of the experiment wherein after 30-minute pre-incubation with 50μg/ml of the protein synthesis inhibitor cycloheximide, MCF-7 cells weretreated with vehicle (DMSO) or 100 μM IB-MECA for the indicated hours(hr). Cells were harvested and assayed for ERα or actin contents withWestern blot analyses. FIG. 8C shows the ERα mRNA half-life in IB-MECAtreated cells. MCF-7 cells were pre-treated with vehicle (DMSO, −) or100 μM IB-MECA (+) for 6 hours before adding the transcription inhibitorDRB (80 μM). Cells were harvested after indicated periods, and weresubjected to RT-PCR analyses using specific primers for ERα. Total RNAsamples of 2 μg each were resolved on a denaturing agarose gel and the18S rRNA bands were used as loading controls. FIG. 8D demonstratesresults from a representative experiment illustrateing the linear rangeof PCR reactions. Indicated template amounts of the 0 hour DMSO-treatedsample in (FIG. 8C) were amplified. A representative picture of PCRproducts analyzed on an acrylamide gel is shown. Intensities of thebands were quantitated using Kodak Digital Scientific 1D software andpresented in arbitrary units (AU). Data shown are averages of two PCRreactions and error bars represent variations. A linear regressionfitting curve was plotted with R² value of 1. FIG. 8E shows that mRNAhalf-lives as quantitated for samples in (FIG. 8C). Average intensitiesof duplicate PCR reaction products (for ERα) were normalized withcorresponding intensities of 18S rRNA. Normalized ERα data werepresented as the percentage of the level at 0 hour time point, andplotted on a logarithmic scale. Data shown are averages of threeindependent experiments and error bars represent standard deviations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating individuals havingmalignancies associated with estrogen receptor activity comprisingadministering to an individual affected with said malignancy, aneffective amount of adenosine analog in a pharmaceutical carrier todownregulate or diminish estrogen receptors in the cells. Preferably,the malignancy is breast cancer or ovarian cancer.

The invention is based upon a surprising finding that adenosine analogsdiminish, or downregulate the amount of estrogen receptors and estrogendependent cancer cell growth.

In a preferred embodiment, the adenosine analog is selected from thegroup consisting of N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide(IB-MECA), 2-chloro-deoxy-adenosine (CdA), 3′-deoxyadenosine(Cordycepin), 2-chloro-N-6-cyclopentyladenosine (CCPA),5′-(N-Ethylcarboxamido) adenosine (NECA), 2-chloro-adenosine (CADO),inosine (INO), which all can be purchased from Sigma (St. Louis, Mo.).More preferably, the adenosine analog is an A3 adenosine receptorselective analog, for example, IB-MECA.

The term “treatment” as used throughout the specification means: (1)preventing such disease from occurring in a subject who may bepredisposed to these diseases but who has not yet been diagnosed ashaving them; (2) inhibiting these diseases, i.e., arresting or slowingdown their development; or (3) ameliorating or relieving the symptoms ofthese diseases.

The term “effective amount” as used throughout the specification meansan amount of the compound necessary to obtain a detectable therapeuticeffect. The therapeutic effect may include, for example, withoutlimitation, inhibiting the growth of undesired tissue or malignantcells, inhibition of tumor cell growth, decreased levels of an estrogenreceptor transcript or protein. Estrogen receptors include estrogenreceptor alpha (or ESR1, OMIM ID No. 13340, athttp://www.ncbi.nlm.nih.gov) and estrogen receptor beta (or ESR2, OMIMID No. 601663, at http://www.ncbi.nlm.nih.gov), and the like. Theprecise effective amount for a subject will depend upon the subject'ssize and health, the nature and severity of the condition to be treated,and the like. Thus, it is not possible to specify an exact effectiveamount in advance. However, the effective amount for a given situationcan be determined by routine experimentation based on the informationprovided herein.

Individuals who can be treated with the methods of the present inventioninclude those affected with an estrogen receptor associated cancersincluding osteosarcomas, pituitary adenomas, testicular, uterine,ovarian and breast cancers. Different types of breast cancers include,but are not limited to ductal carcinoma in situ (DCIS), infiltrating (orinvasive) ductal carcinoma (IDC), or infiltrating (or invasive) lobularcarcinoma (ILC). In one preferred embodiment, the individual is affectedwith breast cancer wherein the cancer cells are estrogen receptor (ER)positive. In one preferred embodiment, the ER is ERalpha. One preferredgroup of individuals treated by the present invention are those havingtumors containing cells that exhibit anchorage independent growth.

In another preferred embodiment, the individual affected with breastcancer which is unresponsive to tamoxifen, 4-OH-tamoxifen, raloxifene,or ICI 164,384 therapy.

For therapeutic applications, the compounds may be suitably administeredto the individual affected with cancer, alone or as part of apharmaceutical composition, comprising the compounds together with oneor more acceptable carriers thereof and optionally other therapeuticingredients. The carrier(s) must be “pharmaceutically acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. In one embodiment, theadenosine analog of the present invention is administered together withtamoxifen, 4-OH-tamoxifen, raloxifene, or ICI 164,384, or a mixturethereof.

The pharmaceutical compositions of the present invention include thosesuitable for oral, rectal, nasal, (including buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenousand intradermal), occular using eye drops, transpulmonary usingaerosolubilized or nebulized drug administration. The formulations mayconveniently be presented in unit dosage form, e.g., tablets andsustained release capsules, and in liposomes, and may be prepared by anymethods well know in the art of pharmacy. (See, for example, Remington:The Science and Practice of Pharmacy by Alfonso R. Gennaro (Ed.) 20thedition, Dec. 15, 2000, Lippincott, Williams & Wilkins; ISBN:0683306472.)

When preparing the pharmaceutical composition of the present invention,such preparative methods include the step of bringing into associationwith the adenosine analog or a derivative thereof ingredients such asthe carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing into association the active ingredients, including theadenosine analogs, with liquid carriers, liposomes or finely dividedsolid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion, or packed in liposomes and as a bolus,etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets optionally may be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

It will be appreciated that actual preferred amounts of a given compoundused in a given therapy will vary according to the particular adenosineanalog compound being utilized, the particular compositions formulated,the mode of application, the particular site of administration, thepatient's weight, general health, sex, etc., the particular indicationbeing treated, etc. and other such factors that are recognized by thoseskilled in the art including the attendant physician. Optimaladministration rates for a given protocol of administration can bereadily determined by those skilled in the art using conventional dosagedetermination tests.

In one embodiment, the invention provides a pharmaceutical compositionfor suppressing cell cycle and/or cellular growth comprising aneffective amount of at least one adenosine analog and a pharmaceuticallyacceptable carrier. In one preferred embodiment, the adenosine analog isselected from the group consisting of A3 receptor binding analog,IB-MECA, 2-chloro-adenosine, and estrogen receptor downregulatingderivatives thereof.

A method of identifying ER inhibitory compounds, including adenosineanalogs, useful for the treatment of cancer, such as breast or ovariancancer, comprises the steps of treating cancer cells with the adenosineanalog in question and calculating cell growth, measuring ERalpha levelsby western blot analysis and/or quantitative RT-PCR, and determiningcell cycle arrest by flow cytometry analysis.

In another preferred embodiment, this treatment is combined with anotherform of cancer therapy including use of SERMS such as tamoxifen,radiation, a chemotherapeutic, an antiangiogenic agent, etc.Anti-angiogeneic agents are known to one skilled in the art and include,but are not limited to VEGF and its receptors (Kim et al., Nature362:841-844, 1993; Saleh et al., Cancer Res 56:393-401, 1996; Millaueret al., Cancer Res 56:1615-1620, 1996; Millauer et al., Nature367:576-579, 1994; Strawn et al., Cancer Res 56:3540-3545, 1996; VEGFantagonists (Claffey et al., Cancer Res 56:172-181, 1996); both humanand murine forms of angiostatin, a proteolytic fragment of plasminogen(O'Reilly et al., Cell 79:315-28, 1994; O'Reilly et al., Nat Med2:689-92, 1996). Similarly, a C-terminal fragment of collagen XVIII,termed endostatin, has been reported to exhibit anti-angiogenic andtumor-regressing activities accompanied by a lack of acquired tumorresistance (O'Reilly et al., Cell 88:277-85, 1997; Boehm et al., Nature390:404-7, 1997); and vector-mediated delivery of angiostatin,endostatin, soluble Flt1 ectodomains, and soluble neuropilin (sNRP)domains, (see, e.g., Takayama et al., Cancer Res 60:2169-77, 2000;Griscelli et al., Proc Natl Acad Sci USA 95:6367-6372, 1998; Blezingeret al., Nat Biotechnol 17:343-8 1999; Chen et al., Cancer Res59:3308-3312, 1999; Sauter et al., Proc Natl Acad Sci USA 97:4802-4807,2000; Feldman et al., Cancer Res 60:1503-1506, 2000).

The invention further provides a use of pharmaceutical compoundscomprising adenosine analogs, such as A3 adenosine receptor agonists,IB-MECA, 2-chloro-adenosine and derivatives thereof, for treatment ofcancer. The cancer preferably comprises cells expressing estrogenreceptors, most preferably ERalpha. The most preferred treatment targetsare breast cancer and ovarian cancer. In one embodiment, the cancercomprises cells growing anchorage independently.

The present invention also provides kits for detecting or screeningcancer treatment compounds capable of downregulating estrogen receptors.Such kits typically comprise two or more components necessary forperforming a screening assay of compounds that are capable ofdownregulating estrogen receptors and therefore useful in treatment ofcancers. Components may be compounds, cells, reagents, containers and/orequipment. For example, one container within a kit may contain amonoclonal antibody or fragment thereof that specifically binds estrogenreceptor to enable detection of downregulation of estrogen receptors inthe cells. Such antibodies or fragments may be provided attached to asupport materials known to one skilled in the art. One or moreadditional containers may enclose elements, such as reagents or buffers,to be used in the assay. Such kits may also, or alternatively, contain adetection reagent as described above that contains a reporter groupsuitable for direct or indirect detection of antibody binding.

In one preferred embodiment, the kit is designed to detect and measureestrogen receptor mRNA level. Such kits generally comprise at least oneoligonucleotide probe or primer, that hybridizes to a polynucleotideencoding estrogen receptor protein(s). Such an oligonucleotide may beused, for example, within a reverse transcriptase (RT)-PCR, PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding anestrogen receptor protein. Primers may also be labeled to enhancedetection.

The kits provided by the present invention also include at least onecontrol reagent, such as IB-MECA, or other adenosine analogdownregulating estrogen receptors. Such control reagent is provided sothat it can be administered to the cells expressing estrogen receptorsprovided in the kit, and thereby allow comparison of test compound(s) toan effective estrogen receptor downregulating agent, and consequentlyprovide a reference point for effectiveness of the novel test compoundin downregulating estrogen receptors. The kit also provides instructionshow to measure estrogen receptor downregulation, for example, asprovided by the examples shown in this specification.

Means for detecting estrogen receptor downregulation include, forexample immunological techniques using estrogen receptor antibodies.Preferably, the detection means include techniques based on detection ofmRNA levels such as RT-PCR based methods include, but are not limited toPYROSEQUENCING™ (Uppsala, Sweden); real-time PCR systems which rely uponthe detection and quantitation of a fluorescent reporter, the signal ofwhich increases in direct proportion to the amount of PCR product in areaction, for example TaqMan® (ABI 7700 (TaqMan®), Applied BioSystems,Foster City, Calif.); hybridization-based techniques; an INVADER® assay(Third Wave Technologies, Inc (Madison, Wis.)), fluorescence-based PCRquantification techniques, solid-phase minisequencing (U.S. Pat. No.6,013,431 and in Wartiovaara and Syvanen, Quantitative analysis of humanDNA sequences by PCR and solid-phase minisequencing. Mol Biotechnol 2000June; 15(2):123-131); and MALDI-TOF mass array (Sequenom's MassArray™system).

Test compounds may include small organic or inorganic molecules,libraries of molecules, phage display libraries and the like known toone skilled in the art. For, example, synthetic compound libraries arecommercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Inaddition, natural and synthetically produced libraries can be produced,if desired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modification within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

EXAMPLE

We have found that IB-MECA, an A₃AR agonist, can potently inhibit cellproliferation in both anchorage-independent and anchorage-dependentassays. Our results indicated that the effect of IB-MECA in ERα-positivebreast cancer cells was not mediated by the activation of A₃AR, butrather involved ERα downregulation. These results point to the potentialuse of IB-MECA and its derivaties in the treatment of estrogen receptorpositive cancers, and demonstrate the existence of a signaling pathwayinitiated by IB-MECA and its derivatives, that can regulate ERα andERα-mediated processes.

Methods

Chemicals: All chemicals were purchased from Sigma (St Louis, Mo.),unless otherwise indicated.N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) was purchasedfrom Sigma or from Tocris (Avonmouth, UK), in order to examine twodifferent batches of preparation. IB-MECA,2-Chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (C1-IB-MECA),5′-(N-Ethylcarboxamido)adenosine (NECA) and2-Chloro-N⁶-cyclopentyladenosine (CCPA) were dissolved in DMSO, with astock concentration of 50 mM, and aliquoted and stored in −80° C.Adenosine was freshly dissolved before experiments into whole cellculture medium.2-p-(2-Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine(CGS21680) was dissolved in phosphate buffered saline (PBS) (Invitrogen,Carlsbad, Calif.) at 2 mM.

Plasmids: pERE-Tk-Luc, consisting of a promoter containing estrogenresponsive elements, driving the luciferase reporter gene (39), andpcDNA3-ERα, consisting of the CMV promoter driving the expression ofhuman estrogen receptor cDNA (39) were kind gifts from Dr. ZhixiongXiao. pcDNA3 and pEGFP-C1 plasmids were purchased from Clontech (PaloAlto, Calif.). pRc-hA3AR, consisting of the CMV promoter driving theexpression of the human A3 adenosine receptor cDNA and pCMV-β-Gal,consisting of the CMV promoter driving the bacterial β-Galactosidasegene, were constructed in our lab and verified by DNA sequencing

Cell culture: MCF-7, ZR75 and T47D cells were originally from AmericanType Culture Collection (ATCC) and cultured in Dulbecco's ModifiedEagle's Medium (DMEM, Invitrogen, Carlsbad, Calif.) supplemented with10% fetal bovine serum (FBS), 5 U/ml penicillin, 5 μg/ml streptomycin,and 2 mM L-glutamine (All from Invitrogen, Carlsbad, Calif.). Hs578tcells were cultured in the above medium supplemented with 0.01 mg/mlinsulin (Sigma, St. Louis, Mo.). When indicated, MCF-7 cells werecultured in DMEM medium free of phenol red (Invitrogen, Carlsbad,Calif.) with charcoal stripped serum (Hyclone, Logan, Utah) for 3 daysbefore being treated with drugs.

Anchorage-independent growth (soft agar) assay: Soft agar assay wasperformed as described (40) with the following modifications. Ligandswere added into the bottom and top agar before plating into 6-wellplates. Cells were treated with trypsin (Invitrogen, Carlsbad, Calif.)for 5 minutes in a 37° C. incubator and pipetted several times so thatmost cells were in single cell forms. Cells were counted with ahemacytometer (Hausser Scientific/VWR, So. Plainfield, N.J.), and 10,000cells were mixed with top agar and plated into each well. After the topagar had solidified, two ml of medium containing the same treatment wasadded on top of the agar. This covering medium was changed every twodays during culture. After two weeks of culture, each well was countedfor the number of colonies formed on an Olympus IX70 microscope under40× optical amplification. A cell colony was defined as any cluster ofcells that contain more than 3 cells. The average of counts from 3random optical fields for each well was taken as the colony number andanalyzed. Each treatment was performed in triplicates. The averages andstandard deviations shown in the figures were calculated based ontriplicate experiments.

Anchorage-dependent growth assay: Cells were plated into 6-well platesand grown overnight before treatments. The seeding concentration ofMCF-7 cells was 2×10⁵/well, which was determined during preliminaryexperiments as not allowing the cells to reach confluency within 3 days.Cells were treated either with vehicle or ligands, as indicated. Cellswere detached by incubation with trypsin (Invitrogen, Carlsbad, Calif.)and counted with a hemacytometer before or after treatment.

Western Blot Analysis: Cells were washed three times in cold 1×PBS, andcollected by scraping on ice. Western blot analysis was performed as wedescribed before with the chemiluminescence method (41). Antibodies usedin this study were: ERα (NeoMarkers, Ab-15), Cyclin A (Santa CruzBiotech, H-432), Cyclin B1 (Santa Cruz Biotech, H-433), Cyclin E(Upstate Biotech, HE-12), p27 (Santa Cruz Biotech, F-8). ERα antibodywas used at 1:100 dilutions. Cyclin A, cyclin B1 and p27 antibodies wereused at 1:500 dilutions. Cyclin E antibody was used at 1:1000 dilutions.

Flow Cytometry and Apoptosis Analysis: Cells were detached from tissueculture plates by trypsin treatment. Cells were collected bycentrifugation at 1200 g for 5 minutes and washed once with PBS.Staining of cells with propidium iodide and analysis on a flow cytometer(FACScan, Becton Dickinson, Research Triangle Park, N.C.) was performedas described before (42). Data were analyzed with CellQuest software(Becton Dickinson, Research Triangle Park, N.C.). The percentage ofcells appearing with a ploidy level smaller than a diploid content wascalculated as an estimate of cells undergoing apoptosis.

Transfections and Reporter Gene Assay: Transient transfection wasperformed using FuGene6 (Roche, Indianapolis, Ind.) transfection reagentaccording to manufacturer's protocol. Circular reporter plasmidpERE-Tk-Luc at 10 μg and 10 μg of pCMV-β-Gal (as a measure of efficiencyof trasnfection) were transfected with 50 μl of FuGene6. Cells weresplit into 6 well plates 12 hours after transfection, and incubatedovernight in fresh medium. Cells were treated with vehicle or 100 μMIB-MECA for 12 hours before harvesting. Luciferase and β-galactosidaseactivities were measured as described before (43, 44).

Stable transfection was performed with similar procedures as transienttransfection, except that the plasmid pRc-A3AR was linearized with PvuI,and purified by phenol/chloroform extraction and ethanol precipitation.Transfected MCF-7 cells were selected with 500 μg/ml of Geneticin(Invitrogen, Carlsbad, Calif.), until Geneticin treated control cellsall died. This pool of stably transfected cells were either used inexperiments, or subjected to single clone selection with limiteddilution as described before (43). Briefly, cells were diluted into aconcentration of 2.5 cells per ml, and added into 96 well plates at 200μl/well. Clones of cells grown up were analyzed for their A3ARexpression using RT-PCR.

Total RNA preparation and Reverse Transcription Polymerase ChainReaction (RT-PCR): Total RNA from MCF-7 cells was prepared with Trizol(Invitrogen, Carlsbad, Calif.) as described before (41). For reversetranscription, 2 μg of RNA were used in a 20 μg reaction with randomprimers and M-MLV reverse transcriptase (Invitrogen, Carlsbad, Calif.),following the manufacturer's protocol. To control for possiblecontamination from genomic DNA in subsequent PCR reactions, controlreverse transcription reactions were carried out under identicalconditions, only without reverse transcriptase. After reversetranscription, 5% of the product was used in each PCR reaction. For theexperiments analyzing A3 adenosine receptor (A3AR) expression, 27 cycleswere used in the PCR reactions. Specific primers were designed for humanA3AR, which match to two separate exons, according to genomic sequences(from GenBank). Sequences for the sense and antisense A3AR primers are:5′tccatcatgtccttgctg3′ (SEQ ID NO: 1) and 5′gcacatgacaaccaggg3′ (SEQ IDNO.: 2). In the experiments analyzing estrogen receptor a (ERα) mRNA,semi-quantitative PCR reactions were carried out with 23 cycles for ERαprimers and 19 cycles for GAPDH primers (used as a control). The cyclenumbers were tested in previous experiments not to produce saturationeffects. The sense and antisense primer sequences for estrogen receptora are: 5′gatccaagggaacgagctgg3′ (SEQ ID NO.: 3) and5′tgggctcgttctccaggtag3′ (SEQ ID NO.: 4). The sense and antisense primersequences for GAPDH are: 5′tcaccatcttccaggag3′ (SEQ ID NO.: 5) and5′gcttcaccaccttcttg3′ (SEQ ID NO.: 6).

Thymidine Incorporation Assay. Thymidine incorporation assays wereperformed as described (Zhang, Y., Wang, Z., and Ravid, K. The cellcycle in polyploid megakaryocytes is associated with reduced activity ofcyclin B1-dependent cdc2 kinase. J Biol Chem, 271: 4266-4272, 1996) withmodifications. Rat bone marrow cells were cultured in 25 cm² flasks at aconcentration of 20×10⁶ cells per 2 ml. After drug treatment for 24hours, cells were incubated with ³H-thymidine at a final concentrationof 3 μCi/ml for 8 hours. For MCF-7 cells, cells were cultured in 6-wellplates and incubated with ³H-thymidine for 2 hours after drug treatment.Cells were divided into two portions, sixty percent of which wereprocessed as described (Id.) to obtain tritium counts. The rest of thecells were lysed with Western blotting lysis buffer and proteinconcentrations were determined by Bio-Rad protein assay reagent(Bio-Rad, Hercules, Calif.). Tritium counts were normalized withcorresponding protein concentrations to account for cell numbervariations.

Messenger RNA Half-life Determination. MCF-7 cells were pretreated withvehicle (DMSO) or 100 μM IB-MECA for 6 hours, followed by addition of 80μM of DRB (5,6-dichlorobenzimidazole riboside) or 50 μM of actinomycinD. Cells were harvested either before addition of transcriptioninhibitor (0 hour) or after different time periods. Total RNA wasprepared and ERα content was assayed by RT-PCR analyses as described inthe methods for RT-PCR. To control for the amount of RNA used in reversetranscription reactions, 2 μg each of total RNA were resolved on adenaturing agarose gel as described before (Cataldo, L. M., Zhang, Y.,Lu, J., and Ravid, K. Rat NAP1: cDNA cloning and upregulation by Mp1ligand. Gene, 226: 355-364, 1999) and stained with ethidium bromide.

Results

Adenosine or IB-MECA inhibits anchorage-independent growth of MCF-7cells. It has been reported that skeletal muscle-conditioned medium,with adenosine as an active component, can inhibit anchorage-dependentgrowth of MCF-7 breast cancer cells, as measured by thymidineincorporation (15). We examined whether adenosine can also inhibit theanchorage-independent growth of MCF-7 cells, a hallmark oftumorogenesis, and if this effect was mimicked by adenosine analogs.Adenosine was added into soft agar cultures at different concentrations,and colonies formed were counted after two weeks of culturing. As shownin FIG. 1A, adenosine displayed a dose-dependent inhibition of colonyformation. At 1 mM, adenosine inhibited approximately 50% of thecolony-forming ability of MCF-7 cells. No effect was observed wheninosine was used instead of adenosine (not shown).

Such a high concentration of adenosine can hardly be achieved duringnormal physiological processes. Since adenosine exerts many of itseffects through the activation of adenosine receptors and many adenosinereceptor agonists have a higher stability than adenosine, we askedwhether agonists for the four types of adenosine receptors could inhibitanchorage-independent growth of MCF-7 cells. CCPA (A1AR agonist), NECA(A2AR agonist), CGS21680 (A2aAR agonist), and IB-MECA (A3AR agonist)were used at different concentrations. At much higher concentrationsthan their binding affinities, CCPA, NECA, and CGS21680 did not inhibitthe anchorage-independent growth of MCF-7 cells (FIG. 1B through 1D).IB-MECA, on the other hand, at concentrations from 10 to 100 μM, showeda dose-dependent inhibition of MCF-7 cell colony formation (FIG. 1E).

Effects of IB-MECA on anchorage-independent growth, anchorage-dependentgrowth and apoptosis of different breast cancer cell lines: We testedthe effect of IB-MECA on several human breast cancer cell lines,including ZR-75, T47D (ERα positive), Hs578T (ERα negative) and HeLa(human cervix adenocarcinoma cell line). All breast cancer cell linestested showed a dramatic decrease in colony formation, while HeLa cellsonly exhibited a mild response to this agonist (FIG. 2A), suggestingthat inhibition of anchorage-independent growth by IB-MECA is closelyrelated to the origin of cancer.

The effect of IB-MECA on anchorage-dependent growth was also examined inthese breast cancer cell lines. After three days of treatment inculture, trypan blue negative cells were counted and compared to thecell counts on day 0 (before treatment). Inhibition ofanchorage-dependent growth Was observed with all four breast cancer celllines tested, namely MCF-7, ZR-75, T47D, and Hs578T. The cell countsafter three days of treatment were all lower than those at day 0.Noticeably, however, cell counts of MCF-7 and ZR-75 cells decreased onlymildly while T47D and Hs578T cells were affected more severely (FIG.2B).

Some studies involving examination of mechanisms of IB-MECA effects ongrowth of a variety of transformed cells concluded that increasedapoptosis is involved (13, 14). We examined whether the fraction ofapoptotic cells was increased in IB-MECA-treated breast cancer cells. Wehave elected a quantitative approach to follow apoptotic cells. To thisend, cells were stained with propidium iodide after ligand treatment,and subjected to flow cytometry analyses. The fraction of events withfluorescence intensity less than a diploid DNA content would indicatethe relative population of apoptotic cells. As shown in FIG. 2C, T47Dand Hs578T cells treated with IB-MECA underwent substantial apoptosiscompared to the vehicle-treated samples, while MCF-7 and T47D cellsdisplayed a non significant change in apoptotic events.

These results indicated that IB-MECA can induce two types of signalingin breast cancer cells. One involves growth inhibition and anotherinduces apoptosis. IB-MECA-induced growth arrest in ER positive breastcancer cells, however, has never been reported, and this study willfocus on elucidating the mechanisms of such an effect.

IB-MECA inhibits anchorage-dependent proliferation of MCF-7 cells: SinceIB-MECA inhibited the anchorage-independent proliferation of MCF-7 cellson both colony numbers and sizes we further tested this chemical on theanchorage-dependent proliferation of these cells. The numbers oftrypan-blue negative cells were followed after MCF-7 cells were treatedwith IB-MECA. Although vehicle treated cells showed an exponentialincrease in cell count, cells treated with IB-MECA did not show muchchange in the number of viable cells, even after 3 days of drugtreatment (FIG. 3A). Our data indicated that IB-MECA was able to rapidlyinhibit anchorage-dependent proliferation of MCF-7 cells.

We further tested this inhibition by analyzing DNA synthesis throughthymidine incorporation. Because many chemicals that inhibit cancer cellproliferation have undesirable side-effects on bone marrow cells, wealso tested the effect of IB-MECA on a primary rat bone marrow culturethrough thymidine incorporation. Since bone marrow cells have much lowerrates of proliferation after long periods in culture (not shown) and theeffect of IB-MECA on MCF-7 cells could be observed after 1 day, wetreated the cells for 24 hours before incubating them with thymidine.IB-MECA and 2-chloro-2′-deoxyadenosine (2CdA, a drug used inchemotherapy) decreased thymidine incorporation in MCF-7 cells to 28%and 43% respectively (FIG. 3B). In contrast, IB-MECA at 100 μM had amilder effect on thymidine incorporation in primary bone marrow cells(reduced to 68%), compared to the effect of 2CdA (reduced to 32%) (FIG.3C). Interestingly, in vivo application of IB-MECA had no inhibitoryeffect on blood cell counts, probably due to cytokine influences(Fishman, P., Bar-Yehuda, S., Madi, L., and Cohn, I. A3 adenosinereceptor as a target for cancer therapy. Anticancer Drugs, 13: 437-443,2002).

IB-MECA arrests MCF-7 cells at G1 or G1/S phase of the cell cycle. Wethen explored which point of the cell cycle was blocked by treatment ofIB-MECA. Flow cytometry analysis of MCF-7 cells treated with IB-MECA,compared to vehicle-treated cells, showed that there was a decrease ofS-phase population from 25% to 12% (FIG. 3B). The peak with diploid DNAcontent increased from 50% to 64% after IB-MECA treatment. There wasalso a minute decrease in the population of tetraploid DNA content from24% to 23%. These results suggested that IB-MECA has a primary effect onthe G1/S cell cycle transition.

To further analyze the cell cycle arrest, Western blot analyses werecarried out with antibodies against cyclins and Cdk inhibitors. As shownin FIG. 3C, cyclin A, and B1 were downregulated in MCF-7 cells.Consistent with the previous growth inhibition data, HeLa cells showedno significant change in cyclin levels (data not shown). The decrease incyclins A and B1 was accompanied by a sharp increase in the cdkinhibitor p27. Cyclin E levels were elevated upon ligand treatment, asmight be expected from cell cycle arrest at G1 phase. These dataconfirmed that the cell cycle inhibition was primarily at G1/S.Interestingly, treating MCF-7 cells with different concentrations ofIB-MECA showed decreases in cyclins A and B with a similar dosageresponse as the decrease in anchorage-independent growth (FIG. 3D).

Overexpression of A3AR does not increase the sensitivity of MCF-7 cellstowards IB-MECA treatment: IB-MECA is an A3AR selective agonist. Cellgrowth inhibition by IB-MECA in several transformed cell lines has beenattributed to A3AR activation (31-33). The affinity of IB-MECA for A3adenosine receptor was reported to be in the nanomolar range (34).However, the cell growth inhibitory effect we report here could onlybeen observed at concentrations higher than about 10 μM. There might twopossible reasons that could explain why the concentration needed forgrowth inhibition is much greater than the binding affinity. Onepossibility is that the growth inhibition is not mediated through theA3AR. The second possibility is that MCF-7 cells have low abundanceor/and low affinity A3AR, so that only a high concentration of IB-MECAcan activate a relevant downstream signaling. Indeed, MCF-7 only had avery low level of A3AR expression (FIG. 4A), as endogenous A3AR mRNAcould barely be detected after 33 cycles of RT-PCR reactions (data notshown). If the growth inhibition by IB-MECA was mediated by low levelexpression of A3AR in MCF-7 cells, overexpression of the human A3ARwould increase the sensitivity of cells upon IB-MECA treatment. Toexplore this possibility, MCF-7 cells were stably transfected with humanA3AR cDNA. Expression of A3AR in a stable transfection pool could bestrongly detected with 27 cycles of RT-PCR reactions (FIG. 4A), and wasstronger than the expression level in the brain, where A3AR isabundantly expressed (data not shown). The percentage of cells in thetransfection pool that contain the transgene was estimated by analyzingsingle clones selected from the pool of cells. 16 out of 17 clonesshowed strong increased expression of A3AR (data not shown), verifyingthat majority of the cells within the transfection pool overexpressedA3AR. The pool of stably transfected cells were compared to normal MCF-7cells in soft agar assays. FIG. 4B shows that the two types of cellshave almost identical dosage response to IB-MECA. Increased expressiondid not lower the concentration of IB-MECA needed to induce growthsuppression. In accordance, the selective A3AR antagonist MRS1191 usedat a concentration of up to 1 μM (greater than its Ki) did not abolishIB-MECA inhibitory effect on growth of MCF-7 cells (not shown). Thus, weconcluded that the growth inhibition by IB-MECA is not mediated throughA3AR.

IB-MECA treatment decreases estrogen receptor a in MCF-7 cells: The datadescribed above indicated that IB-MECA inhibits cell cycle progressionprimarily at the G1/S transition. Since estrogen receptor activation isknown to promote cell cycle progression though G1/S and enhance bothanchorage-dependent and anchorage-independent growth of breast cancercells, we asked whether ERα is a primary target of IB-MECA. To this end,the expression of endogenous ERα mRNA was analyzed withsemi-quantitative RT-PCR in MCF-7 cells treated with IB-MECA. A decreasein the expression of ERα could be consistently detected at 6 hours postIB-MECA treatment, compared to GAPDH expression (FIG. 5A). A dramaticdecrease of ERα could be detected after 12 hours and 24 hours oftreatment. This result shows that IB-MECA treatment can either reducetranscription driven by the ERα promoter gene or affect the stability ofERα mRNA. Detailed mechanism of this downregulation is underinvestigation.

As a consequence of a downregulation of ERα mRNA, ERα protein shouldalso show a time-dependent decrease in IB-MECA-treated cells. Indeed,Western blot analyses indicated that ERα protein in MCF-7 cellsdecreased after IB-MECA treatment, and this decrease occurred before thereduction in cyclin levels (FIG. 5B), suggesting that ERα downregulationcould be the reason for cell cycle inhibition. Reduction of ERα proteinlevels was also observed in ZR-75 and T47D cells treated with IB-MECA(not shown), suggesting that the impact of IB-MECA on this protein iscommon to ERα-positive breast cancer cell lines. Using differentconcentrations of IB-MECA, ERα showed a dosage response very similar tothe one of cyclins and the growth inhibition (FIG. 5C). In contrast, nosignificant change of ERα was detected in cells treated with NECA (FIG.5C). These results further suggested that the decrease of ERα might beresponsible for the growth inhibition effect of IB-MECA. Differentbatches of IB-MECA and cell culture medium might have had an impact onthe rate of ERα downregulation. However, this decrease could always bedetected, between 4 and 8 hours post ligand treatment, under both normalculturing condition and with phenol-red free cell culture medium andcharcoal stripped serum, as well as with two different batches ofIB-MECA (data not shown).

Since activators of estrogen receptors, such as 17-β-estrodiol, canreduce ERα level by regulating ERα protein stability, a decrease ofprotein may not correlate with a decrease of ERα activity (Wijayaratne,A. L. and McDonnell, D. P. The human estrogen receptor-alpha is aubiquitinated protein whose stability is affected differentially byagonists, antagonists, and selective estrogen receptor modulators. JBiol Chem, 276: 35684-35692, 2001; Borras, M., Laios, I., el Khissiin,A., Seo, H. S., Lempereur, F., Legros, N., and Leclercq, G. Estrogenicand antiestrogenic regulation of the half-life of covalently labeledestrogen receptor in MCF-7 breast cancer cells. J Steroid Biochem MolBiol, 57: 203-213, 1996). We next examined the transcriptional activityof endogenous ERα after IB-MECA treatment, using a reporter constructcontaining estrogen responsive elements (EREs) in the promoter. Shown inFIG. 5D, after 12 hours of IB-MECA treatment, the ERE promoter activitydropped by more than 5 fold. In contrast, the non-tissue specific CMVpromoter did not show any decrease in activity. These results indicatedthat IB-MECA downregulated ERα and subsequently caused a reducedactivity of this transcription factor.

Overexpression of ERα can reverse the growth inhibition induced byIB-MECA treatment: Results from the above experiments indicate that thegrowth inhibition of IB-MECA is mediated through a decrease in ERα. Toverify this hypothesis, we took advantage of the fact that the CMVpromoter is not affected much by IB-MECA treatment, and hencetransfected cells with ERα cDNA under the control of the CMV promoter.This would provide the cells with abundance of ERα. Indeed, ERα levelswere high in the transfected cells, even after IB-MECA treatment (FIG.6A).

If the inhibition of IB-MECA was mediated through a decrease in ERα, wewould expect to see a moderate or no effect of IB-MECA on growth of ERαoverexpressing cells. Since only cells transfected with ERα may exhibitany resistance, transfection efficiency will be key in determining thepotential increase in cell counts as compared to control non-transfectedcells. Stable transfection of ERα was attempted twice without success,suggesting a potential long term harmful effect of high levels of ERα inMCF-7 cells. Instead, we overexpressed ERα by transient transfection,and a transfection efficiency of approximately 40% was determined bycounting green cells from a parallel transfection with a CMV-drivengreen fluorescence protein construct (pEGFP-C1).

When ERα was overexpressed in MCF-7 cells, IB-MECA treatment resulted ina moderate effect on growth, as compared to a larger effect in controlcells (FIG. 6B). It is reasonable to assume that IB-MECA effect ongrowth of the transfected pool of cells was not eliminated because ofonly approximately 40% of the cells overexpressed the transgene.

Following methods of assays described above, we analyzed the ability ofother adenosine analogs and of the nucleosides adenosine and inosine inrespect to the ability to inhibit the growth of MCF-7 cells and affectERα protein levels. Table 1 summarizes the data obtained. Inosine aswell as the A1AR selective analog, CCPA, or the A2-type selective analogNECA had no significant effect on cell growth. We also examinedadenosine analogs, which are not selective for adenosine receptors andhave been described as inhibitors of cancer cells. For example,2-chloro-deoxyadenosine was used in trials for treating chroniclymphocytic leukemia (45), or infantile myofibromatosis (46), and3′-deoxyadenosine was shown to inhibit leukemia cells (47). In ourstudies, 2-chloror-deoxyadenosine significantly inhibited the growth ofMCF-7 human breast cancer cells. In contrast to IB-MECA, however, it wasas effective at 1 μM (not shown) as at 100 μM and it did not have aprominent effect on ERα levels, but induced cellular apoptosis.2-chloro-adenosine>3′-deoxyadenosine significantly inhibited cell growthand ERα levels, without inducing apoptosis. These compounds, as IB-MECAwere only effective when used at a 10-100 micromolar range. They arelikely, however, to act on a different mechanism than IB-MECA becausethey affected the cell cycle at a different phase, i.e., not at G1/S asIB-MECA did. Hence, their reducing effect on ER (which is attenuated ascompared to the effect of IB-MECA) might be a result of a primary effecton cell cycle arrest. These data show that IB-MECA, 2-chloro-adenosineas well as 3′-deoxyadenosine can be used in vivo for inhibition ofbreast cancer. 3′-deoxyadenosine (Cordycepin) was used before inClinical Trials to inhibit specific blood cell cancers.

IB-MECA-induced downregulation of ERα is likely die to decreasedtranscription from the estrogen receptor a gene. Studies on ERαregulation have revealed that this gene is regulated at the levels oftranscription (McPherson, L. A., Baichwal, V. R., and Weigel, R. J.Identification of ERF-1 as a member of the AP2 transcription factorfamily. Proc Natl Acad Sci USA, 94: 4342-4347, 1997), mRNA stability(Saceda, M., Lindsey, R. K., Solomon, H., Angeloni, S. V., and Martin,M. B. Estradiol regulates estrogen receptor mRNA stability. J SteroidBiochem Mol Biol, 66: 113-120, 1998; Ing, N. H. and Ott, T. L. Estradiolup-regulates estrogen receptor-alpha messenger ribonucleic acid in sheependometrium by increasing its stability. Biol Reprod, 60: 134-139, 1999;Kenealy, M. R., Flouriot, G., Sonntag-Buck, V., Dandekar, T., Brand, H.,and Gamnon, F. The 3′-untranslated region of the human estrogen receptoralpha gene mediates rapid messenger ribonucleic acid turnover.Endocrinology, 141: 2805-2813, 2000), and protein degradation(Wijayaratne, A. L. and McDonnell, D. P. The human estrogenreceptor-alpha is a ubiquitinated protein whose stability is affecteddifferentially by agonists, antagonists, and selective estrogen receptormodulators. J Biol Chem, 276: 35684-35692, 2001; Borras, M., Laios, I.,el Khissiin, A., Seo, H. S., Lempereur, F., Legros, N., and Leclercq, G.Estrogenic and antiestrogenic regulation of the half-life of covalentlylabeled estrogen receptor in MCF-7 breast cancer cells. J SteroidBiochem Mol Biol, 57: 203-213, 1996). We investigated the downregulationinduced by IB-MECA by first examining the abundance of ERα mRNA. Forcomparison between protein levels and mRNA levels, materials from thesame samples as in FIG. 6B were used for total RNA preparation. As shownin FIG. 7A, IB-MECA strongly downregulated ERα mRNA in a fast andtime-dependent manner, with the first sign of decrease after 4 hours ofIB-MECA treatment. Following ERα downregulation, the mRNA level of pS2,an endogenous estrogen-responsive gene (55), was also reduced byIB-MECA. The downregulation of pS2 could be observed after 8 to 12 hours(FIG. 7A), and to a stronger degree after 24 hours (not shown). Incontrast, the message level of another estrogen binding protein,estrogen receptor β (56), was not significantly reduced (FIG. 7A). ThiscDNA was amplified with primers corresponding to the first two exons ofthe estrogen receptor β gene. ERα mRNA downregulation precedes that ofERα protein (FIGS. 6B and 8A), suggesting that the primary effect ofIB-MECA is on ERα mRNA. Indeed, we did not notice any significant changein ERα protein degradation upon IB-MECA treatment, when proteinsynthesis was inhibited by cycloheximide (FIG. 8B).

To differentiate whether the effect was on ERα gene transcription ormRNA stability, we examined the half-life of ERα message in vehicle- orIB-MECA-treated cells. MCF-7 cells were pretreated with vehicle orIB-MECA for 6 hours before adding the transcription inhibitor5,6-dichlorobenzimidazole riboside (DRB) which causes prematuretranscription termination. Consistent with FIG. 8A, ERα mRNA wasdecreased after 6 hours of IB-MECA pretreatment, as revealed bysemi-quantitative RT-PCR (the 0 hour time point, FIG. 8C). The PCRreactions were carried out under conditions that allow linearamplification and quantitation (FIG. 8D). The half-life of ERα measuredunder the experimental conditions may be longer than the real half-lifein the cells, since the used transcription inhibitor may not shut downtranscription immediately. Nevertheless, comparing vehicle- andIB-MECA-treated cells would indicate whether there is an impressivedifference in ERα half-lives. The mRNA half-life in IB-MECA-treatedcells was similar to that in vehicle-treated cells (FIGS. 8C and 8E),and the difference could not account for the observed reduction in mRNAlevel. Inhibiting transcription with another transcription inhibitor,actinomycin D, showed similar results (not shown). Thus, we concludedthat the effect of IB-MECA on ERα was most likely on the transcriptionof the gene. It should be pointed out that nuclear run-on assays wereattempted, as we described before (Wang, Z., Zhang, Y., Lu, J., Sun, S.,and Ravid, K. Mp1 ligand enhances the transcription of the cyclin D3gene: a potential role for Sp1 transcription factor. Blood, 93:4208-4221, 1999), but ERα de novo transcription in control cells wasbelow our detection limit.

Discussion

While adenosine and chemically-synthesized adenosine receptor agonistshave been reported to inhibit cancer cell proliferation, theseinhibitory effects are through various mechanisms, and mainly via theactivation of different adenosine receptors. In contrast, found thathigh concentrations of adenosine inhibited growth of cancers havinganchorage-independent cells such as MCF-7 breast cancer cells. Among theagonists examined in our study, IB-MECA was shown to be a potent growthinhibitor of breast cancer cell lines, while the A₁AR agonist, CCPA, andthe A₂AR agonists, CGS21680 and NECA, did not have a significant effecton MCF-7 cell proliferation. The breast cancer cells examined showed nodetectible levels of A₃AR, and A₃AR overexpression in MCF-7 cells didnot result in increased sensitivity upon IB-MECA treatment. In addition,an A₃AR antagonist did not abolish the effect of IB-MECA. This suggestedthat A₃AR is not a primary pathway through which the growth inhibitionis mediated. Another A₃AR agonist, chloro-IB-MECA, was shown to induceapoptosis in two leukemia cell lines, through mechanisms not related toA₃AR signaling (Kim, S. G., Ravi, G., Hoffmann, C., Jung, Y. J., Kim,M., Chen, A., and Jacobson, K. A. p53-Independent induction of Fas andapoptosis in leukemic cells by an adenosine derivative, C1-IB-MECA.Biochem Pharmacol, 63: 871-880, 2002).

How IB-MECA triggers the effect on proliferation in the treated breastcancer cells is not clear. It is possible that IB-MECA at highconcentrations binds other unidentified membrane receptors and triggersdownstream signaling. Another possibility could be that the compoundsignals through direct interaction with intracellular targets, afterbeing transported into the cell. Such intracellular mechanisms have beennoticed for adenosine (Schrier, S. M., van Tilburg, E. W., van derMeulen, H., Ijzerman, A. P., Mulder, G. J., and Nagelkerke, J. F.Extracellular adenosine-induced apoptosis in mouse neuroblastoma cells:studies on involvement of adenosine receptors and adenosine uptake.Biochem Pharmacol, 61: 417-425, 2001) and an adenosine analog,2-chloroadenosine (Barbieri, D., Abbracchio, M. P., Salvioli, S., Monti,D., Cossarizza, A., Ceruti, S., Brambilla, R., Cattabeni, F., Jacobson,K. A., and Franceschi, C. Apoptosis by 2-chloro-2′-deoxy-adenosine and2-chloro-adenosine in human peripheral blood mononuclear cells.Neurochem Int, 32: 493-504, 1998), using the nucleoside transporterinhibitor dipyridamole. In our system, 10 μM dipyridamole did notprevent the growth inhibitory effect of IB-MECA, while at higherconcentrations, dipyridamole had by itself an inhibitory effect on cellgrowth. It is possible, however, that the nucleoside uptake inhibitorcan not fully block the transport of IB-MECA, since IB-MECA at higherconcentrations might compete for the transporters or enter the cell by anucleoside transporter-independent mechanism. In lack of radio-labeledIB-MECA, we were not able to determine the intracellular concentrationof this ligand. The details of the mechanisms by which IB-MECA targetsits effecter molecules are intriguing and await further exploration.

In search for mechanisms of action of IB-MECA on breast cancer cellgrowth, we focused on a known regulator of these cells, the estrogenreceptor α. We showed that, in ERα-positive breast cancer cell linesMCF-7, ZR-75, and T47, IB-MECA downregulated ERα, suggesting that thiseffect is general in ERα-positive breast cancer cells. We also showedthat reversing the downregulation of ERα significantly attenuated thegrowth inhibition induced by IB-MECA, indicating that ERα downregulationis one pathway responsible for the growth inhibition in ER-positivebreast cancer cells. This, however, does not exclude the possibilitythat other pathways are also involved in IB-MECA-induced proliferationinhibition in these cells. We found that IB-MECA regulated ERα through adownregulation of mRNA and protein, and consequently ERα atranscriptional activity. The half-life of ERα message was notsignificantly altered when IB-MECA was present. This eliminates thepossibility of regulation on message stability and points to a highlikelihood of regulation through gene transcription. The ERα genecontains multiple promoters, some of which are as far as 150 kb upstreamof the primary transcriptional start site (Kos, M., Reid, G., Denger,S., and Gannon, F. Minireview: genomic organization of the human ERalphagene promoter region. Mol Endocrinol, 15: 2057-2063, 2001; Reid, G.,Denger, S., Kos, M., and Gannon, F. Human estrogen receptor-alpha:regulation by synthesis, modification and degradation. Cell Mol LifeSci, 59: 821-831, 2002). Only a few transcription factors are known toregulate ERα gene expression (McPherson, L. A., Baichwal, V. R., andWeigel, R. J. Identification of ERF-1 as a member of the AP2transcription factor family. Proc Natl Acad Sci USA, 94: 4342-4347,1997; Cohn, C. S., Sullivan, J. A., Kiefer, T., and Hill, S. M.Identification of an enhancer element in the estrogen receptor upstreamregion: implications for regulation of ER transcription in breastcancer. Mol Cell Endocrinol, 158: 25-36, 1999), including AP2γ. Furtherexperiments are needed to elucidate the detailed mechanism of ERα genedownregulation by IB-MECA. The mechanism by which IB-MECA downregulatesERα is different from the one found for selective estrogen receptordownregulators, such as ICI 182,780 (also known as fulvestrant andFaslodex). ICI 182,780 reduces ERα level through increased ERα proteinturnover (Wakeling, A. E., Dukes, M., and Bowler, J. A potent specificpure antiestrogen with clinical potential. Cancer Res, 51: 3867-3873,1991; Pink, J. J. and Jordan, V. C. Models of estrogen receptorregulation by estrogens and antiestrogens in breast cancer cell lines.Cancer Res, 56: 2321-2330, 1996; Fan, M., Bigsby, R. M., and Nephew, K.P. The NEDD8 pathway is required for proteasome-mediated degradation ofhuman estrogen receptor (ER)-alpha and essential for theantiproliferative activity of ICI 182,780 in ERalpha-positive breastcancer cells. Mol Endocrinol, 17: 356-365, 2003), while IB-MECAdownregulates ERα through an effect on gene expression. In this view,IB-MECA and similar compounds may be efficacious in the treatment ofbreast cancers that are resistant to or have acquired resistance(Lykkesfeldt, A. E., Larsen, S. S., and Briand, P. Human breast cancercell lines resistant to pure anti-estrogens are sensitive to tamoxifentreatment. Int J Cancer, 61: 529-534, 1995) to the pure antiestrogen ICI182,780, and thus might be important additions to the arsenal ofendocrine therapies for human breast cancer.

We examined the effect of IB-MECA on several different breast cancercell lines. IB-MECA inhibited the growth of MCF-7 and ZR-75 cells, andinduced apoptosis in T47D and Hs578T cells. In T47D cells, IB-MECAtreatment downregulated estrogen receptor a in a similar manner as inMCF-7 cells. It is known that T47D cells areestrogen-signaling-dependent; estrogen stimulates the proliferation ofT47D cells, and inhibiting estrogen signaling results in an inhibitionof proliferation (Jones, J. L., Daley, B. J., Enderson, B. L., Zhou, J.R., and Karlstad, M. D. Genistein inhibits tamoxifen effects on cellproliferation and cell cycle arrest in T47D breast cancer cells. AmSurg, 68: 575-577; discussion 577-578, 2002; Fontana, J. A. Interactionof retinoids and tamoxifen on the inhibition of human mammary carcinomacell proliferation. Exp Cell Biol, 55: 136-144, 1987; Dardes, R. C.,O'Regan, R. M., Gajdos, C., Robinson, S. P., Bentrem, D., De Los Reyes,A., and Jordan, V. C. Effects of a new clinically relevant antiestrogen(GW5638) related to tamoxifen on breast and endometrial cancer growth invivo. Clin Cancer Res, 8: 1995-2001, 2002; Cavailles, V., Gompel, A.,Portois, M. C., Thenot, S., Mabon, N., and Vignon, F. Comparativeactivity of pulsed or continuous estradiol exposure on gene expressionand proliferation of normal and tumoral human breast cells. J MolEndocrinol, 28: 165-175, 2002). Thus, it is possible that in T47D cells,two different pathways were induced by IB-MECA. One pathway involves ERαwhich is common in all ERα-positive cells and which would lead toproliferation inhibition. Another pathway, which is not activated inMCF-7 cells and ZR-75 cells, initiates apoptosis in T47D cells. In MCF-7cells, IB-MECA does not induce or inhibit apoptosis. Apoptotic eventscan be initiated via a variety of signaling pathways and by activationof one or more related regulators (reviewed in Green, D. R. and Reed, J.C. Mitochondria and apoptosis. Science, 281: 1309-1312, 1998; Wajant, H.The Fas signaling pathway: more than a paradigm. Science, 296:1635-1636, 2002; Vousden, K. H. p53: death star. Cell, 103: 691-694,2000). We speculate that IB-MECA does not initiate apoptosis in MCF-7cells because of its ability to activate certain anti-apoptoticmolecules, such as Akt (reviewed in Franke, T. F., Kaplan, D. R., andCantley, L. C. PI3K: downstream AKTion blocks apoptosis. Cell, 88:435-437, 1997; Datta, S. R., Brunet, A., and Greenberg, M. E. Cellularsurvival: a play in three Akts. Genes Dev, 13: 2905-2927, 1999) so thatthe balance between its apoptotic and anti-apoptotic signals aremaintained. Hence, the dominant effect of IB-MECA in MCF-7 cells isinhibition of ERα expression and proliferation. We found that IB-MECAinduced Akt phosphorylation (at Ser 473) in MCF-7 cells (not shown), asalso reported in rat basophilic leukemia 2H3 cells (Gao, Z., Li, B. S.,Day, Y. J., and Linden, J. A3 adenosine receptor activation triggersphosphorylation of protein kinase B and protects rat basophilic leukemia2H3 mast cells from apoptosis. Mol Pharmacol, 59: 76-82, 2001). Thisdoes not imply, however, that Akt is the only pathway by which IB-MECAmight affect apoptosis in these cells. Further exploration is needed toreveal the detailed mechanisms by which apoptosis is induced by IB-MECAin some cell lines, but not in others.

In summary, we made the novel findings that IB-MECA potently inhibits ERpositive cancer cell growth via downregulation of ERα, rather thanthrough A3AR signaling. This shows that IB-MECA, and its functionalderivatives can be used as a drug to treat patients with cancersexpressing estrogen receptors, such as breast cancer. It may also beused in therapies that are aimed at regulating ERα levels. A variety ofother adenosine analogs might be screened for inhibition of growth ofbreast cancer cells in vitro, using the tools we employed here. TABLE 1Effects of Different Adenosine Analogues on the Growth of the BreastCancer Cell Line MCF-7 Compound¹ Cell Growth² Apoptosis³ ERα Level⁴ CellCycle Arrest⁵ Control (Vehicle) 1 1 1 None N⁶-(3- −0.19 ± 0.06   1.12 ±0.02 0.23 G1/S iodobenzyl)adenosine- (28, −51, −5) 5′-N- methyluronamide(IB-MECA) 2-chloro-deoxy- 0.13 ± 0.01 1.80 ± 0.17 0.85 S adenosine (CdA)(−69, 165, 20) 3′-deoxyadenosine 0.24 ± 6   0.43 ± 0.07 0.58 G2/M(Cordycepin) (−26, 10, 62) 2-chloro-N⁶- 0.79 ± 0.16 0.49 ± 0.12 1.16G2/M cyclopentyladenosine (3, −18, 11) (CCPA) 5′-(N- 0.90 ± 0.16 0.99 ±0.05 0.92 Not Detectable Ethylcarboxami- (0, 3, −3) do)adenosine (NECA)2-chloro-adenosine −0.02 ± 0.02   0.84 ± 0.6  0.41 G2/M and S (CADO)(−11, 11, 20) Adenosine 0.49 ± 0.04 1.06 ± 0.17 1.07 G2/M (ADO) (−25,−56, 97) Inosine 0.90 ± 0.12 1.02 ± 0.14 ND S (INO) (−24, 43, 8)¹Compounds were used at 100 μM, except for adenosine (500 μM) andinosine (1 mM).²Viable cell numbers were determined by staining with Trypan Blue andcounting on a hematocytometer. Cells were counted after 2 days oftreament and cell growth was calculated, normalizing to vehicle control(arbitrarily set to 1). A negative number means a decrease of cellnumber compared to cell count right before treatment. Data shown areaverages ± standard deviations from three experiments.³Data represent the number of apoptotic cells after 2 days of treatmentcompared to vehicle control, determined as described under Methods. Invehicle control, 10% to 20% of the cells were apoptotic and werearbitrarily set to 1. Data are averages ± standard deviations from threeexperiments.⁴Cells were treated with indicated drugs for 12 hours and ERα proteinlevel was measured by Western blot analyses, and quantitated using KodakDigital Scientific 1D software. The ERα level of vehicle control wasarbitrarily set to 1, and data represent the averages of twoexperiments.⁵Cell cycle arrest was determined by flowcytometry analyses. Proportionsof cells with diploid (G0 or G1 phase), tetraploid (G2 or M phase) orintermediate-state (S phase) DNA contents were compared to those ofvehicle-treated cells. Cell cycle phases were used to designate theposition of cell cycle arrest. Percentage changes of diploid, S,tetraploid populations, compared to those of vehicle-treated cells, werelisted in parentheses.ND: Not Determined

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1. A method for downregulating estrogen receptors in a population ofcells expressing an estrogen receptor comprising delivering to saidpopulation of cells an effective amount of at least one adenosine analogand a pharmaceutically acceptable carrier to down-regulate estrogenreceptor levels.
 2. The method of claim 1, wherein the population ofcells comprise malignant cells.
 3. The method of claim 2, wherein themalignant cells are breast cancer cells.
 4. The method of claim 2 or 3,wherein the population of cells are estrogen receptor alpha positive. 5.The method of claim 1, wherein the adenosine analog is an adenosine A3receptor agonist.
 6. The method of claim 5, wherein the adenosine analogis N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide or a derivativethereof.
 7. The method of claim 5, wherein the adenosine analog is2-chloro-adenosine or a derivative thereof.
 8. The method of claim 1,wherein the downregulation of estrogen receptor levels results fromdecrease in estrogen receptor transcription.
 9. The method of claim 1,wherein at least one cell in the population of cells is or has becomeresistant to (Z)1,2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene, 4-OH-(Z)1,2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene, raloxifene, orN-(n-butyl)-1-[3,17β-dihydroxyestra-1,3,5(10)-trien-7α-yl]N—methylundecanamide or a derivative thereof.
 10. The method of claim 1,wherein the at least one adenosine analog and a pharmaceuticallyacceptable carrier to decrease estrogen receptor levels are deliveredbefore, after or simultaneously with (Z)1,2-diphenyl-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1-butene, 4-OH-(Z)1,2-diphenyl-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1-butene,raloxifene, orN-(n-butyl)-1′-[3,17β-dihydroxyestra-1,3,5(10)-trien-7α-yl]N—methylundecanamide or other estrogen receptor regulating pharmaceutical,or a combination thereof.
 11. The method of claim 1, wherein at leastone cell in the population of cells is growing via anchorage-independentmanner.
 12. The method of claim 1, wherein at least one cell in thepopulation of cells is growing via anchorage-dependent manner.
 13. Themethod of claim 4, wherein the population of cells comprise at least onecell which is estrogen receptor alpha positive.
 14. A method ofsuppressing cell cycle and/or cellular growth in a population of cellscomprising delivering to the cell population an effective amount todownregulate estrogen receptor levels, at least one adenosine analog anda pharmaceutically acceptable carrier.
 15. The method of claim 14,wherein the population of cells comprises malignant cells.
 16. Themethod of claim 15, wherein the malignant cells are breast cancer cells.17. The method of claim 15, wherein the malignant cells are ovariancancer cells.
 18. A method of treating an individual affected withmalignant cell growth in a tissue or plurality of tissues expressingestrogen receptors, the method comprising administering to theindividual a sufficient amount of an adenosine agonist to downregulateestrogen receptors in a cell population in the tissue or plurality oftissues and a pharmaceutically acceptable carrier.
 19. The method ofclaim 18, wherein the malignant cell growth is breast cancer.
 20. Themethod of claim 18, wherein the malignant cell growth is ovarian cancer.21. The method of claim 18, wherein the malignant cell growth isanchorage-independent.
 22. The method of claim 18, wherein at least oneof the estrogen receptors expressed by the cell population is mutated ortruncated.
 23. The method of claim 18, wherein the estrogen receptor isestrogen receptor alpha.
 24. The method of claim 23, wherein themalignant cell growth is breast cancer or ovarian cancer.
 25. A methodof identifying a compound suitable for treating malignant cell growth ina tissue which expresses estrogen receptors, the method comprisingmeasuring the amount of estrogen receptor expression in a cell,administering an adenosine analog to the cell, and measuring theexpression of estrogen receptor after administration of the adenosineanalogue, wherein reduction in the amount of estrogen receptor in thecell after administration of the adenosine analogue indicatesidentification of a compound suitable for treating malignant cellgrowth.
 26. The method of claim 25, wherein the malignant cell growth isbeast cancer or ovarian cancer. 27-32. (canceled)
 33. A kit fordownregulating estrogen receptors in a population of breast and/orovarian cancer cells comprising in a container at least one adenosineanalog capable of downregulating estrogen receptors, wherein theadenosine analog is an adenosine A3 receptor agonist selected from thegroup consisting of N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide,2-chloro-adenosine and a derivative ofN⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide, 2-chloro-adenosine, inthe population of cells in a pharmaceutically acceptable carrier in avial or tube, a means for detecting downregulation of estrogen receptorsin the population of cells, and an instruction manual exemplifying howto measure estrogen receptor downregulation using the means provided inthe kit. 34-36. (canceled)
 37. A kit for detecting compounds capable ofdownregulating estrogen receptors in a population of cells comprising: apopulation of test cells expressing estrogen receptors in a suitablecell growth medium or freezing medium or storage medium; a standardadenosine analog capable of downregulating estrogen receptors in thepopulation of test cells as powder or in a suitable buffer with knownconcentration; a means for detecting estrogen downregulation in the testcell population, wherein the test cell population comprises malignantcells, wherein the malignant cells are breast and/or ovarian cancercells and wherein the malignant cells are resistant to a compoundselected from the group consisting of (Z)1,2-diphenyl-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1-butene, 4-OH-(Z)1,2-diphenyl-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1-butene,raloxifene, orN-(n-butyl)-11-[3,17β-dihydroxyestra-1,3,5(10)-trien-7α-yl]N-methylundecanamide;and an instruction manual outlining exemplary cell growth conditions todetect downregulation of estrogen receptors in the test cell populationusing the standard adenosine analog, wherein the standard adenosineanalog is selected from the group consisting of adenosine A3 receptoragonist, N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide and2-chloro-adenosine. 38-47. (canceled)